Antibacterial and cytotoxic activities of naphthoquinone pigments from Onosma visianii Clem

In this study, the antibacterial and cytotoxic activities of isolated compounds from the roots of Onosma visianii were investigated. By using different chromatographic techniques and appropriate spectroscopic methods, the seven naphthoquinones were described: deoxyshikonin (1), isobutyrylshikonin (2), α-methylbutyrylshikonin (3), acetylshikonin (4), β-hydroxyisovalerylshikonin (5), 5,8-O-dimethyl isobutyrylshikonin (6) and 5,8-O-dimethyl deoxyshikonin (7). Among the tested compounds, 3 and 4 exhibited the highest antibacterial activities toward all tested bacterial species (MIC50 and MIC90 for gram positive bacteria: 6.40 μg/mL-12.79 μg/mL and 6.82 μg/mL-13.60 μg/mL, respectively; for gram negative bacteria: 4.27 μg/mL-8.53 μg/mL and 4.77 μg/mL-9.54 μg/mL, respectively). Also, naphthoquinones 3 and 4 exhibited strong cytotoxic activity against MDA-MB-231 cells (IC50 values 86.0 μg/mL and 80.2 μg/mL, respectively), while compounds 1, 3, 4 and 5 significantly decreased viability of HCT116 cells (IC50 values of 97.8 μg/mL, 15.2 μg/mL, 24.6 μg/mL and 30.9 μg/mL, respectively). Our results indicated that all tested naphthoquinone pigments are potential candidates for clinical uses as antibacterial and cytotoxic agents.


INTRODUCTION
The emergence of antibiotic resistance presents one of the most important challenges for the pharmaceutical industry and the healthcare sector both in the developing and developed countries. The inadequate and excessive use of antibiotics, significantly diminishes the efficacy of current drugs, at the same time demanding new drugs against multi-drug resistant bacteria (Gyles, 2011). Another major health problem is cancer. It affects about two hundred types of cell lines, causing lack of control of the cell proliferation and differentiation while invading various tissues and organs. There are many difficulties in the treatment of these diseases, especially in terms of drug resistance, toxicity, and low specificity. Although the official medicine has shown effective results, herbal medicinal products successfully act on some kinds of these ailments.
The genus Onosma L. (Boraginaceae) contains about 150 species, distributed mainly in the Mediterranean region and Central Asia (El-Shazly et al., 2003).
Phytochemical investigation of the roots of genus Onosma has led to the discovery of a series of shikonin and alkannin derivatives, as compounds which exhibit a wide spectrum of biological properties, like wound healing, antiinflammatory, antitumor, antimicrobial and antithrombotic properties (Papageorgiou et al., 1999(Papageorgiou et al., , 2008Wang et al., 2015;Naz et al., 2006). On the other hand, literature reported that aerial parts contain pyrrolizidine alkaloids (El-Shazly et al., 2003;Damianakos et al., 2013) and phenolic compounds (Mellidis et al., 1993).
Onosma visianii Clem (Boraginaceae) is a biennial to perennial plant which inhabits areas of Balkan Peninsula and Southeast Europe. The point of view of the chemical composition, O. visianii is little investigated. Except that it contains shikonin (Shcherbanovskii, 1971), other literature data relating to chemical composition and pharmacological activity do not exist.
We herein report for the first time isolation and structural characterization of these naphthoquinones from the roots of O. visianii. Also, antibacterial activity against ten bacterial species, as well as cytotoxic activity against MDA-MB-231 and HCT116 tumor cell lines was described.

Instrumentation
Microanalysis of carbon and hydrogen was carried out with a Carlo Erba 1106 microanalyser. Infrared spectra were run on Thermo Scientific Nicolet 6700 FT-IR spectrometer (4000-400 cm -1 ) and Perkin-Elmer FT-IR Spectrometer Spectrum One (KBr disc,  in cm -1 ). The NMR spectra were recorded on a Varian Gemini 200 spectrometer ( 1 H at 200 MHz and 13 C at 50 MHz) and a BrukerAvance III 500 spectrometer ( 1 H at 500.26 MHz and 13 C at 125.80 MHz), solvent CDCl3, TMS internal standard. Chemical shifts were given in  (ppm), J-coupling constants in Hertz (Hz), abbreviations: s-singlet, d-doublet, dd-doublet of doublet, t-triplet, mmultiplet, br s-broadend singlet. Low resolution mass spectra were recorded on an Agilent 5973 mass spectrometer (Santa Clara, CA, USA). High resolution mass spectra were obtained on a Agilent 6210 Time of Flight (TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and linear-trap quadrupole (LTQ) orbitrap hybrid mass spectrometer equipped with a heated-electrospray ionization probe (HESI-II, ThermoFisher Scientific, Bremen, Germany).

Extraction method
Dried roots (135 g) of O. visianii were powdered and extracted at room temperature three times with a (1500 mL, 24 h) petroleum ether-methylene chloride mixture (1:1). The combined extracts were evaporated under reduced pressure to give concentrated extract as red-brown semi-viscous residue (6.5 g).

Bacteria
Antibacterial activity was tested against five gram positive bacteria (Bacillus megaterium, Enterococcus faecalis, Microbacterium arborescens, Micrococcus luteus and Staphylococcus epidermidis) and five gram negative bacteria (Citrobacter koseri, Hafnia alvei, Pseudomonas proteolytica, Stenotrophomonas maltophilia and Yersinia intermedia). The tested bacteria (as clinical isolates; confirmed via MALDI TOF MS Biotyper, Bruker Daltonics, Billerica, USA) were provided from a collection of Department of Microbiology, Faculty of Biotechnology and Food Sciences, Nitra, Slovakia.

Suspension preparation
All microorganisms were kept on -80 °C. Cultures of bacteria were incubated on Mueller Hinton agar (Merck, Germany) for 24 h at 36 °C. Regulation of initial suspension turbidity was conducted by comparison with 0.5 McFarland's standard (Andrews, 2005). Initial bacterial suspensions contained about 10 8 CFU/mL. In addition, 1:100 dilutions of initial suspension were made into sterile 0.85 % NaCl.

Micro-dilution antibacterial assay
Minimal inhibitory concentrations (MICs) were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute document (CLSI, 2009) in Mueller Hinton broth (Biolife, Italy). Briefly, the DMSO solutions of isolated naphthoquinones (or standard antibiotics) were prepared as serial two-fold dilutions with the final concentrations ranging between 0.25-512 g/mL. After that, each well was inoculated with microbial suspension at the final density of 0.5 McFarland. After 24 h of incubation at 37 °C, the inhibition of microbial growth was evaluated by measuring the well absorbance at 450 nm in an absorbance microplate reader Biotek EL808 with shaker (Biotek Instruments, USA). The 96microtiter plates were read before and after experiment. Measurement error was established for 0.05 values of absorbance. Wells without tested naphthoquinones (or standard antibiotics) were used as negative controls of growth. Pure DMSO was used as a negative control. This experiment was done in eightreplicates for a higher accuracy of the MICs of tested compounds.

Statistical analysis for antibacterial activity
Differences in absorbance between the measurements before and after the analysis were expressed as a set of binary values. These values were assigned to exact concentrations. The following formula was created for this specific experiment: value 1 (inhibitory effect) was assigned to absorbance values lower than 0.05, while value 0 (no effect or stimulant effect) was assigned to absorbance values higher than 0.05. For this statistical evaluation the probit analysis in Statgraphics software was used.

Drugs and chemicals
Tested isolated naphthoquinones were diluted in DMSO at the concentration of 200 g/mL, filtered through a 0.22 m Millipore filter before use, and diluted by a cell culture medium to diverse working concentrations, so that the final concentration of DMSO in cell culture medium never exceeded 0.5 % (v/v).

Cell lines
Human breast carcinoma cell line MDA-MB-231 and colon cancer cell line HCT116 were purchased from the American Type Culture Collection (ATCC, USA) and cultured as suggested by the supplier. Both cell lines were sustained in culture medium supplemented with 10 % heat-inactivated FBS.

MTT cell viability assay
MDA-MB-231 and HCT116 cells were plated onto microtiter plates at density of 5 x 10 4 /mL and allowed to adhere overnight. Cells were then treated with tested substances at indicated concentrations, or with media alone (control). The plates were incubated for 24, 48 and 72 h at 37 °C in an atmosphere of 5 % CO2 and absolute humidity. The media was removed and 100 L of MTT (0.5 mg/mL PBS) was added to each well. After 4h incubation at 37 °C, MTT solution was removed and 150 L of DMSO was added to dissolve the formazan crystals. Absorbance was measured with a multiplate reader (Zenith 3100, Anthos Labtec Instruments GmbH, Austria) at 595 nm (Mosmann, 1983). The IC50 values were calculated using Microsoft Office Excel free add-in (ed50v10.xls.) for linear regression downloaded from http://www.sciencegateway.org/protocols/cellbio/drug/data/.

Cell cycle analysis
MDA-MB-231 and HCT116 cells were incubated with media alone (control) or with 100 g/mL of compounds 1-5 and 500 g/mL of compounds 6 and 7. After 48 h at 37 °C in an atmosphere of 5 % CO2 and absolute humidity, both attached and detached cells were collected, washed in PBS and fixed with 70 % ethanol at 4 °C. Fixed cells were washed in PBS and finally resuspended in 1mL PBS containing RNAse A (500 g/mL). After 30 min incubation at 37 °C, cells were stained with 5 L PI (10 mg/mL PBS), incubated for 15 min in the dark and immediately analyzed by flow cytometer Cytomics FC500 (Beckman Coulter, USA). Distribution of cells with different DNA content was determined with Flowing Software (http://www.flowingsoftware.com/) and presented by histograms.

Determination of apoptosis/necrosis by flow cytometry
Apoptosis was determined using Annexin V-FITC/7-AAD Kit. MDA-MB-231 and HCT116 cells were incubated with media alone (control) or with 100 g/mL of compounds 1-5 and 500 g/mL of compounds 6 and 7. After 48 h incubation at 37 °C in the atmosphere containing 5 % CO2, both attached and detached cells were collected. After washing with PBS, cells were resuspended in 500 L of ice cold binding buffer. Ten L of Annexin V-FITC and 20 L of 7-AAD were added and after 15 min incubation in the dark, 400 L of binding buffer was added to each tube. Samples were analyzed by flow cytometer Cytomics FC500 (Beckman Coulter, USA). Data were analyzed using Flowing Software and presented by dot plots.
The compounds 1-5 showed IR absorptions in the region of 3680-3160 cm -1 which indicated presence of OH groups attached to naphthoquinone moiety, as well as tertiary OH group from hydroxyisovaleryl part of compound 5. Except in the cases of 1 and 7, observed stretching vibrations of compounds 2-6 in the region of 1742-1732 cm -1 belong to the aliphatic ester C=O groups. Also, for all isolated naphthoquinones (1-7), strong bands from C-O groups were noted (at 1278-1018 cm -1 ). The 1 H NMR spectra of 1-5 showed two deshielded protons from hydroxyl groups attached to C-5 and C-8 carbons from naphthoquinone part (12.41 ppm-12.64 ppm). Also, compound 5 contains one additional broadened singlet (at 3.26 ppm) from hydroxyl group linked to C-3'. Instead of singlets of OH protons, resonances from C-5-OCH3 and C-8-OCH3 groups (singlets at 3.95 ppm and 3.96 ppm, respectively) were observed in the 1 H NMR spectra of 6 and 7 (as dimethoxy derivatives). In contrast to other isolated compounds (for 2-6, doublet of doublets in the region of 6.02 ppm-6.10 ppm), deoxyshikonin 1 and it 5,8-dimethoxy derivative 7 showed signals of C-11-H protons at lower fields (multiplets at 2.28 ppm and 2.52 ppm, respectively). All derivatives contain characteristic triplets from vinyl C-13-H protons (at 5.12 ppm-5.13 ppm), singlets from C-3-H protons (naphthoquinone moiety; at 6.60 ppm-7.03 ppm), as well as singlets from C-H-6 and C-H-7 protons (naphthoquinone moiety; at 7.18 ppm-7.31 ppm). In the 13 C NMR spectra of 2-6, signals from C-1' carbons (at 169.5 ppm-175.7 ppm) were recorded, indicating the presence of aliphatic ester C=O groups. These signals were not observed in cases of compounds 1 and 7, as deoxyshikonin and its dimethoxy derivative. Also, in contrast to isolated compounds 2 and 1, 5,8-dimethoxy derivatives 6 and 7 showed two additional signals from C-5-OCH3 and C-8-OCH3 carbons (56.8 ppm and 56.9 ppm, respectively). Finally, the positive values of measured optical rotations ([]D 25 ) confirmed that isolated naphthoquinones 2-6 from the roots of O. visianii are shikonin derivatives.

In vitro activity of the isolated naphthoquinones against bacteria
The seven isolated naphthoquinones were evaluated for their antibacterial activity against the five gram positive and five gram negative bacteria (Table 1). Resistance of used bacteria was presented in Table 1, while the activities of isolated compounds (as MIC50 and MIC90 in g/mL, respectively) were showed in Table 2. Observed MIC50 and MIC90 values of isolated naphthoquinones are in the range of 4.27 g/mL-68.27 g/mL and 4.77g/mL-76.20g/mL, respectively. In accordance with literature (Noundou et al., 2016), compounds which exhibited MIC values equal or less than 16 g/mL were considered to have possible clinical relevance. Following these facts, erythromycin, ampicillin and vancomycin resistant B. megaterium is the most sensitive bacteria against tested compounds 1, 3 and 4, ampicillin and vancomycin resistant E. faecalis against compounds 3, 4 and 5, while other three gram positive bacteria are sensitive against 3 and 4. Among gram negative bacteria, meropenem resistant S. maltophilia is susceptible to 2, 3, and 4, while other four bacteria showed high sensitivity against compounds 3 and 4. Previously published data indicated notable activity of shikonin derivatives against gram positive bacteria (and generally low activity against gram negative bacteria), with the fact of remarkable differences of antibacterial potency due to different approaches of measuring MIC values (Papageorgiou et al., 1999;Andujar et al., 2013). On the other hand, some results showed good antibacterial activity against some gram negative species (Brigham et al., 1999;Ding et al., 2011). Our results indicated that isolated naphthoquinone derivatives at the same time showed a good activity towards both gram positive and gram negative bacteria.  Among the isolated compounds, αmethylbutyrylshikonin 3 and acetylshikonin 4 exhibited the highest antibacterial activities toward all tested bacterial species. Observed MIC50 and MIC90 values for gram positive bacteria are in the range of 6.40 µg/mL-12.79 μg/mL and 6.82 μ g/mL-13.60 μg/mL, respectively, while for gram negative bacteria are in the range of 4.27 μg/mL-8.53 μg/mL and 4.77 μg/mL-9.54 μg/mL, respectively. The shikonin derivative 3 had strong potency against E. faecalis (MIC50 6.40 μg/mL and MIC90 6.82 μg/mL), as well as against S. maltophilia (MIC50 4.27 μg/mL and MIC90 4.77 µg/mL), while compound 4 was very active against M. arborescens (MIC50 6.40 μg/mL and MIC90 6.82 μg/mL) and P. proteolytica (MIC50 4.27 μg/mL and MIC90 4.7 μg/mL). In contrast to previous report that alkylation of the phenolic groups leads to lower activity (Papageorgiou et al., 1999), we herein reported that gram negative ampicillin resistant bacteria Y. intermedia was very susceptible against compound 7 as dimethoxy derivative of deoxyshikonin (MIC50 12.79 μg/mL and MIC90 13.60 μg/mL).
From the above mentioned results, in cases of developed resistance (or in cases of preventing resistance) examined naphthoquinones may substitute clinical antibiotics.

Cytotoxic activity of isolated naphthoquinones
Cytotoxic activity of compounds 1-7 against two cancer cell lines was performed by conducting MTT cell viability assays. Generally, tested compounds decreased viability of both tumor cell lines in a dose and time dependent manner. Yet, the extent of cytotoxicity on the designated cell lines was diverse, being more potent on HCT116 cell line ( Figure 2, Table 3).
As observed in Table 3, -methylbutyrylshikonin 3 and acetylshikonin 4 exhibited the highest cytotoxic activity against MDA-MB-231 cells compared to all other tested substances with (IC50 values 86.0 g/mL and 80.2 g/mL, respectively). Also, all examined compounds, except compounds 6 and 7, decreased viability of MDA-MB-231 cells after 48 hours of incubation. However, HCT116 cells were generally more sensitive to the effects of isolated naphthoquinones. Compound 3 showed the strongest cytotoxic effect against HCT116 cells with IC50 value of 15.2 g/mL. Also, compounds 1, 4 and 5 significantly decreased viability of HCT116 cells and these compounds exhibited IC50 values of 97.8g/mL, 24.6g/mL and 30.9g/mL, respectively. The results presented in Table 3 are in accordance with the previously published data of cytotoxicity of shikonin derivatives against many cancer cell lines. Among 1500 tested quinones, about 150 compounds showed strong in vivo activity against W256 in rats and P388 lymphoid leukemia in mice, as well as significant in vitro potency toward KB cells and CCRF-CEM leukemia cells (Papageorgiou et al., 1999). On the other hand, isolated quinones demonstrated significant in vitro potency toward KB cells, CCRF-CEM leukemia cells, as well as some melanoma cell lines (Papageorgiou et al., 1999;Kretschmer et al., 2012). To determine whether cytotoxicity induced by naphthoquinones was due to apoptosis or cell cycle arrest, the cells were stained with fluorescent dyes and analyzed on flow cytometer.

Naphthoquinones induce apoptosis in tumor cell lines
Flow cytometric analysis showed that primary type of cell death in MDA-MB231 cells, induced by isolated naphthoquinones, was apoptosis, with small proportion of necrotic cells, whereas percent of necrotic cells was considerable higher only in cells treated with compound 4 (Figure 3).  Similarly, in HCT116 cells, the supreme type of cell death induced by naphthoquinones was apoptosis and again, only compound 4 induced similar percent of both apoptosis and necrosis ( Figure 3). The scope of cancer therapy is to promote apoptosis, a type of cell death that is, unlike necrosis, limited to cells committed to die and does not affect surrounding tissue. Therefore, agents that induce apoptosis in cancer cells can be useful in cancer treatment.

Naphthoquinones induce cell cycle arrest in tumor cell lines
A 48 hour treatment of MDA-MB-231 cells with compounds 3-7 resulted in significant increase in percentage of the cells assembled in G2/M phase, with concomitant decrease in percentage of cells in G0/G1 phase (p < 0.05) (Figure 4).
The percentage of MDA-MB-231 cells batched in G2/M phase increased from 7.6 % in control to 14.0 % -40.8 % in treated cells. Additionally, compared to control, compounds 5, 6 and 7 showed a significant rise in the number of the cells arrested at S phase (3.2 %, 10.1 %, 25.4 % and 27.1 % respectively). Compounds 1 and 2 showed no changes in cell cycle profile of MDA-MB-231 cells (Figure 4). Similarly, treatment of HCT116 cells with compounds 3, 4, 5 and 6 resulted in increase of G2/M phase cells from 30.7 % (control) to 38.9 %-49.6 %, with most pronounced arrest when treated with compound 3 (p < 0.05). Contrary, treatment with compounds 2 and 7 showed an increasing trend in the number of the cells arrested at G0/G1 phase: quantitatively it was 24.3 % and 14.7 % respectively over the control values. Compound 1 showed no changes in cell cycle profile of HCT116 cells (Figure 4 and Figure  5). Physiologically, cell cycle and apoptosis are directly connected. In damaged cells, stopping the cell cycle, gives cells a time to activate repair mechanisms and fix the damage. If the damage can't be fixed, apoptosis program is activated. Contrary, uncontrolled progression of the cell cycle and avoidance of apoptosis are the main characteristics of malignant cells (Hanahan and Weinberg, 2000). Our results demonstrated that naphthoquinones arrest mitosis and make the cell cycle stopped in G0/G1, S or G2/M phases resulting in cellular apoptosis of target cells. The difference between cytotoxic effects of isolated naphthoquinones on selected tumor cell lines, was probably caused by different molecular targets of these compounds (Narang and Desai, 2009). Literature reported that activity of naphthoquinones was closely related to do presence of quinone part, with the facts that mode of cytotoxic action includes oxidative stress, as well as inhibition of topoisomerase I and topoisomerase II (Papageorgiou et al, 1999). Thus, G0/G1 arrest in HCT116 (compounds 2 and 7) or S phase arrest in MDA-MB-231 (compounds 6 and 7) point to blockade of DNA replication and inhibition of topoisomerase II and I as a possible mechanism that triggers apoptosis (Huang et al., 2003), whereas G2/M arrest coupled with decreased expression of cyclin-dependent kinases 1 and 2 with contribution from ROSmediated pathways (Pan et al., 2015) may be responsible for apoptosis of MDA-MB-231 cells induced by compounds 3-7.

CONCLUSIONS
In this study, for the first time we presents that roots of O. visianii are a rich source of bioactive naphthoquinone derivatives which possess strong activity toward selected re-sistant bacterial strains, as well as high cytotoxic activity against MDA-MB-231 and HCT116 tumor cell lines. According to our results,-methylbutyrylshikonin 3 and acetylshikonin 4 showed notable activities toward all tested gram positive and gram negative bacterial species. Also, compounds 3 and 4 exhibited strong cytotoxic potency against MDA-MB-231 cells, while compounds 3, 4 and 5 showed the strongest cytotoxic effect toward HCT116 cells. Overall, this research provided a strong indication of the potential use of O. visianii naphthoquinone pigments as antibacterial and cytotoxic agents.